JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2020-097 e2020-97 | Vol. 52

Description of microulae sp. n. (Nematoda: ) from China  a new cyst in the Goettingiana group

Wenhao Li1, Huixia Li1,*, Chunhui Ni1, Deliang Peng2, Yonggang Liu3, Ning Luo1 and Abstract 1 Xuefen Xu A new cyst-forming nematode, Heterodera microulae sp. n., was 1College of Plant Protection, Gansu isolated from the roots and rhizosphere soil of Microula sikkimensis Agricultural University/Biocontrol in China. Morphologically, the new species is characterized by Engineering Laboratory of Crop lemon-shaped body with an extruded neck and obtuse vulval cone. Diseases and Pests of Gansu The vulval cone of the new species appeared to be ambifenestrate Province, Lanzhou, 730070, without bullae and a weak underbridge. The second-stage juveniles Gansu Province, China. have a longer body length with four lateral lines, strong stylets with rounded and flat stylet knobs, tail with a comparatively longer hyaline 2 State Key Laboratory for Biology area, and a sharp terminus. The phylogenetic analyses based on of Plant Diseases and Insect ITS-rDNA, D2-D3 of 28S rDNA, and COI sequences revealed that the Pests, Institute of Plant Protection, new species formed a separate clade from other Heterodera species Chinese Academy of Agricultural in Goettingiana group, which further support the unique status of Sciences, Beijing, 100193, China. H. microulae sp. n. Therefore, it is described herein as a new species 3Institute of Plant Protection, Gansu of genus Heterodera; additionally, the present study provided the first Academy of Agricultural Sciences, record of Goettingiana group in Gansu Province, China. Lanzhou, 730070, Gansu Province, China. Keywords *E-mail: [email protected] Goettingiana group, Heterodera, Morphology, New species, Phylogeny, . This paper was edited by Thomas Powers. Received for publication July 5, 2020.

Cyst-forming are the economical pests of Due to overlapping morphological characters cultivated crops and known to be reported from all the and phenotypic plasticity, it is difficult to distinguish continents (Jones et al., 2013). The genus Heterodera closely related Heterodera species; therefore, was erected by Schmidt (1871) and currently contains sequence-based diagnosis is gaining more reliability about 80 species (Subbotin et al., 2010). Literature for precise and accurate identification of cyst- studies have indicated the presence of 14 Heterodera forming nematodes (Peng et al., 2003). The internal species from China mainland, including H. avenae transcribed spacer region of the ribosomal DNA (Chen et al., 1991), H. glycines (Liu et al., 1994), (ITS-rDNA), the D2 and D3 expansion fragments of H. sinensis (Chen and Zheng, 1994), H. filipjevi (Li the 28S ribosomal DNA genes (D2-D3 of 28S-rDNA), et al., 2010), H. koreana (Wang et al., 2012; Wang et al., and mitochondrial DNA (COI gene) units are good 2012b), H. elachista (Ding et al., 2012), H. ripae (Wang candidate genes for molecular taxonomic and et al., 2012a; Wang et al., 2012b), H. hainanensis phylogenetic studies (Subbotin et al., 2001; Subbotin (Zhuo et al., 2013), H. fengi (Wang et al., 2013), et al., 2006; Madani et al., 2004; Vovlas et al., 2017). H. guangdongensis (Zhuo et al., 2014), H. zeae Based on morphomolecular characterizations, (Wu et al., 2017), H. sojae (Zhen et al., 2018), Handoo and Subbotin (2018) divided Heterodera into H. schachtii, and H. vallicola (Peng et al., 2020). nine distinct groups such as Afenestrata, Avenae,

© 2020 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Heterodera microulae sp. n. from China: Li et al.

Bifenestra, Cardiolata, Cyperi, Goettingiana, Humuli, The 28S D2-D3 region was amplified with the D2A Sacchari, and Schachtii group. Sequence analysis of (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B these groups is significant to study the phylogenetic (5′-TCGGAAGGAACCAGCTACTA-3′) (De Ley et al., relationship and identifying the Heterodera species. 2005; Ye et al., 2007). Finally, the partial COI gene During 2018 and 2019, a population of cyst was amplified using primers Het-coxiF (5′-TAGTT nematode was collected from the rhizosphere of GATCGTAATTTTAATGG-3′) and Het-coxiR (5′-CCT Microula sikkimensis in Tianzhu county of Gansu AAAACATAATGAAAATGWGC-3′) (Subbotin, 2015). Province, China. Considering the economic value PCR conditions were as described by Ye et al. (2007), of the cyst nematode, morphomolecular studies De Ley et al. (2005), and Subbotin (2015). PCR pro­ were performed; the preliminary studies indicated ducts were separated on 1% agarose gels and that the population belongs to Goettingiana group visualized by staining with ethidium bromide. PCR of Heterodera. The species characters were then products of sufficiently high quality were purified for compared with all the related species and concluded cloning and sequencing by Tsingke Biotech Co. Ltd., that this population possess unique characters Xi’an, China. The PCR products were purified by the and it is described herein as Heterodera microulae Tiangen Gel Extraction Kit (Tiangen Biotech Co. Ltd., sp. n. Beijing, China), cloned into pMD18-T vectors and transformed into DH5α -competent cells, and then Materials and methods sequenced by Tsingke Biotech Co. Ltd (Xi’an, China).

Isolation and morphological observation Sequence alignment and phylogenetic of nematodes analysis The nematodes were extracted from root and soil The newly obtained sequences for each gene (ITS- samples of Microula sikkimensis in Tianzhu county, rDNA, D2-D3 region of 28S-rDNA, and COI gene) Gansu Province, China. Cysts and white females were compared with known sequences of Heterodera were collected using sieving-decanting method, while using BLASTn homology search program. Outgroup second-stage juveniles (J2s) were recovered from taxa for phylogenetic analyses were selected based hatched eggs and kept in water suspension until on the previously published studies (Subbotin et al., further use (Hooper, 1970; Golden, 1990). Males were 2001; Maafi et al., 2003; Mundo-Ocampo et al., not found. For morphometric studies, second-stage 2008; Kang et al., 2016; Madani et al., 2018; Vovlas juveniles were killed by gentle heating, fixed in TAF et al., 2017). The selected sequences were aligned solution (formalin: triethanolamine: water = 7:2:91), and by MAFFT (Kazutaka and Standley, 2013) with default processed to ethanol-glycerin dehydration according parameters and edited using Gblock (Castresana, to Seinhorst (1959) as modified by De Grisse (1969) 2000). Phylogenetic analyses were based on Bayesian and mounted on permanent slides. Vulval cones inference (BI) using MrBayes 3.1.2 (Huelsenbeck and were mostly mounted in glycerin jelly. Measurements Ronquist, 2001). The GTR + I + G model was selected were made on mounted specimens using a Nikon as the best-fit model of DNA evolution for both 28S Eclipse E100 Microscope (Nikon, Tokyo, Japan). D2-D3, ITS, and COI regions using MrModeltest Light micrographs and illustrations were produced version 2.3 (Nylander, 2004), according to the Akaike using a Zeiss Axio Scope A1 microscope (Zeiss, information criterion (AIC). BI analysis for each gene Jena, Germany) equipped with an AxioCam 105 was initiated with a random starting tree and run color camera and Nikon YS 100 with a drawing tube with four Markov chains for 1,000,000 generations. (Nikon, Tokyo, Japan), respectively. The Markov chains were sampled at intervals of 100 generations and the burn-in value was 25%. Two runs Molecular analyses were performed for each analysis. After discarding burn-in samples, the remaining samples were used DNA samples were prepared according to Maria to generate a 50% majority-rule consensus tree. et al. (2018). Three sets of primers (synthesized by Posterior probabilities (PP) were given on appropriate Tsingke Biotech Co. Ltd., Xi’an, China) were used clades. The phylogenetic consensus trees were in the PCR analyses to amplify sequences of the visualized using FigTree v.1.4.3 software (http://tree. ITS, D2-D3 expansion segments of 28S, and COI bio.ed.ac.uk/software/figtree/) (Rambaut, 2016). The gene. The ITS region was amplified with TW81 species in Goettingiana group and their localities, (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-AT hosts, and GenBank accession numbers used in this ATGCTTAAGTTCAGCGGGT-3′) (Maafi et al., 2003). study were presented in Table S1.

2 JOURNAL OF NEMATOLOGY Results brown; remnants of the subcrystalline layer were rarely present. The egg sac was usually absent (Figs. 1G, Systematics 3B, C). The vulval cone was ambifenestrate-like waning crescent moon and separated by a well-developed Heterodera microulae sp. n. (Figures 1–4; Measure­ vulval bridge. The anus area was distinct, bullae were ment Table 1) absent (Figs. 1F, 3D, E). The vulval slit was longer than fenestral width (39.00 vs 37.75 µm); the underbridge Description was weak and often lost during cone preparation. Cyst Female It is lemon-shaped with an obtuse vulval cone, neck extruding, and cuticle thick with an irregular zig-zag The female was lemon-shaped, pearl white, or pale pattern. The color was white to pale to medium yellow in color. It was rarely rounded with a protruding

Figure 1: Line drawing of H. microulae sp. n. A: Anterior region of second-stage juvenile; B: Head of second-stage juvenile; C: Stylet of second-stage juvenile; D: Tail of second-stage juvenile; E: Cyst; F: Fenestration in vulval cone.

3 Heterodera microulae sp. n. from China: Li et al.

Figure 2: Light micrographs of H. microulae sp. n. A: females attached on M. sikkimensis; B: yellow and white females; C: Anterior region of female; D: Vulval region of female (scale bar: A = 2 mm; B = 1 mm; C, D = 20 µm). neck and vulva, the subcrystalline layer was present, was weak, the stylet was strong, and basal knobs and the egg sac absent (Figs. 2A, B, 3A). There was were rounded and anteriorly flattened. The excretory a labial region with two annuli. Labial sclerotization pore was indistinct, median bulb was rounded and

Figure 3: Light micrographs of H. microulae sp. n. A: immature female on the root; B: Cyst; C: Cysts; D-E: Fenestration in vulval cone; F: Egg (scale bar: A, D = 50 µm; B = 100 µm; C = 200 µm; E, F = 20 µm).

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Figure 4: Light micrographs of second-stage juvenile of H. microulae sp. n. A: Entire body; B: Anterior region of; C: Head region; D: Tail region; E: Posterior pharyngeal region arrow showing the position of dorsal gland nucleus; F: Posterior pharyngeal region arrow showing the position of subventral gland nuclei; G: Lateral field; H: Hemizonid; I: Genital primordium; J: Excretory pore (scale bar: A = 100 µ m, B, H, G = 50 µ m, C, D, E, F, I, J = 20 µ m). massive, and other parts of the pharynx were not nucleus and subventral gland nuclei were distinct clearly discernable. There was vulval slit in a cleft on (Fig. 4E, F). Genital primordium situated at 59 to 62% the cone terminus (Fig. 2C, D). of body length behind the anterior end, with two distinct nucleate cells (Fig. 4I). The tail was conoid, gradually tapering to a finely rounded terminus. The Second-stage juvenile hyaline portion was irregularly annulated occupying 50% of tail length. Phasmid was absent (Figs. 1E, 4D). The body was straight or slightly curved ventrally after heat treatment (Fig. 4A). The lip region was offset and rounded, measuring 3.90 to 5.50 (4.63) µm in height Eggs and 9.65 to 12.75 (11.01)-µm wide. The cephalic framework was strongly sclerotized (Figs. 1B, 4C). Body hyaline without any markings was presented; The stylet was strong; knobs were well developed, juveniles folded six times (Fig. 3F). rounded and flat, or slightly concave anteriorly (Figs. 1C, 4C). The dorsal esophageal gland orifice Male measured from 5.32 to 6.32 (5.61) µm posterior to the stylet knob. Median bulb was rounded with a strong The male was not found. valvular apparatus. The pharyngeal glands were well developed, overlapping the intestine dorsoventrally Type material (Figs. 1A, 4B). The hemizoind was distinct from one to three annuli long (Fig. 4H), the excretory pore Holotype and paratype material (20 cysts, 20 females, was situated 102.46 to 130.79 (114.40) µm from the and 20 second-stage juveniles) were deposited in anterior end, and one to two annules were posterior the nematode collection of the Department of Plant to the hemizonid (Fig. 4G). There was a lateral field Protection, Biocontrol Engineering Laboratory of Crop with four incisures (Figs. 1D, 4G). The dorsal gland Diseases and Pests of Gansu Province, Lanzhou, China.

5 Heterodera microulae sp. n. from China: Li et al.

Table 1. Morphometrics of H. microulae sp. n.

Stage Character Holotype Paratype

Cyst n 20 L (excluding length) 521.79 495.50 ± 41.01 (413.93-543.23) Diam. 419.33 384.29 ± 43.30 (304.96-455.51) L/Diam 1.27 1.30 ± 0.09 (1.12-1.45) Fenestral length 30.72 31.14 ± 1.36 (28.33-32.78) Fenestral width 36.16 37.75 ± 1.61 (35.46-40.07) Vulval slit length 35.53 39.00 ± 2.78 (35.16-44.38) Female n 20 Length 454.21 ± 28.32 (381.62-496.48) Width 326.47 ± 31.42 (256.32-421.63) Length/width 1.40 ± 0.12 (1.28-1.56) Second-stage juveniles n 20 Body length 567.73 ± 43.24 (505.62-627.92) Body width at mid-body 23.19 ± 1.31 (20.39-25.43) a 24.54 ± 1.34 (21.97-27.67) b 4.37 ± 0.33 (3.91-5.00) c 10.07 ± 1.18 (8.56-12.52) c′ 4.26 ± 0.33 (3.63-4.90) Lip-region height 4.63 ± 0.44 (3.90-5.50) Lip-region diam. 11.07 ± 0.82 (9.65-12.75) Stylet length 25.73 ± 1.21 (24.07-28.92) Stylet base height 2.53 ± 0.33 (2.04-3.19) Stylet base width 5.30 ± 0.54 (4.39-6.09) Median bulb from the anterior end (MB) 85.57 ± 5.02 (76.37-95.26) Opening of dorsal pharyngeal gland from 5.13 ± 0.72 (4.03-6.41) the stylet base (DGO) Excretory pore from the anterior end (EP) 114.40 ± 6.89 (102.46-130.79) Median bulb width (MBW) 12.33 ± 1.49 (10.51-15.82) Diam. at the anus 13.23 ± 1.00 (11.24-15.26) Tail length 56.67 ± 3.75 (48.90-60.80) Hyaline portion tail 28.63 ± 1.91 (24.29-30.60) L/MB 6.65 ± 0.37 (5.76-7.53) TL/H 1.98 ± 0.10 (1.64-2.08) Egg n 20 Length 111.61 ± 8.02 (100.21-124.65) Width 50.34 ± 6.71 (35.64-71.51) Length/width 2.26 ± 0.38 (1.43-3.26) Note: All measurements are in µ m, and in the form: mean ± standard (range).

6 JOURNAL OF NEMATOLOGY Type host and locality 47 µm), longer J2s body length (568 µm vs 422 µm), stylet knobs rounded and flat or slightly concave Heterodera microulae sp. n. was collected from the anteriorly vs concave anterior face, higher MB value roots and rhizosphere soil of Microula sikkimensis (86 µm vs 66 µm), longer excretory pore distance from Hemsl. (Boraginaceae, Tubiflorae, Metachlamydeae) the anterior end (114 µm vs 99 µm), and longer tail in Tianzhu county of Gansu Province, China. The length (57 µm vs 52 µm). geographical position is N 37°11′46″; E 102°47′6″. The new species differs from H. cruciferae This site was located in continental highland with the by having a bigger size of cysts (495 × 384 µm vs vegetation type of meadow grassland and the soil is 429 × 333 µm), slightly shorter fenestral length (31 µm composed of chernozems. The climatic parameters vs 34 µm), shorter vulval length (39 µm vs 45 µm), of the site include 450 mm of average rainfall and an longer J2s body length (568 µm vs 431 µm), higher approximate −2 air temperature. MB value (86 µm vs 68 µm), longer excretory pore distance from the anterior end (114 µm vs 101 µm), Etymology longer tail length (57 µm vs 50 µm), and longer length of hyaline tail portion (29 µm vs 25 µm). The species is named after the host of its isolation. The new species differs from H. persica by a shorter fenestral length (31 µm vs 47 µm), absence of Diagnosis and relationships bullae (vs present), shorter vulval slit length (39 µm vs 49 µm), longer J2s body length (568 µm vs 440 µm), Heterodera microulae sp. n. is characterized by having stylet knobs (flat or concave anteriorly vs projecting lemon-shaped cysts that have protruding necks and slightly anteriorly, convex posteriorly), longer stylet obtuse vulval cones. The cysts are 414 to 543-µm (26 µm vs 23 µm), higher MB value (86 µm vs 70 µm), long and 305 to 456-µm wide having ambifenestrate longer excretory pore distance from the anterior vulval cone and bullae are absent. Females are white end (114 µm vs 103 µm), longer tail length (57 µm vs in color with a subcrystalline layer. Second-stage 47 µm), and longer length of hyaline tail portion (29 µm juveniles are straight or slightly curved ventrally with vs 24 µm). four incisures in the lateral field. The juveniles are Compared with H. urticae, the new species has a 506 to 628-µm long having strong stylets with well- smaller size of cysts (495 × 384 µm vs 492 × 435 µm), developed rounded stylet knobs, genital primordium vulval cone obtrusive (vs unobtrusive) and absence of situated at 59 to 62% of body length, and tail 49 to egg sac (vs presence), shorter fenestral length (31 µm 61-µm long with a hyaline portion of 24 to 33 µm. vs 38 µm), shorter vulval slit length (39 µm vs 46 µm), Eggs are hyaline without any markings; juveniles longer J2s body length (568 µm vs 541 µm), shorter inside the eggs form sixfold. DGO (8 µm vs 5 µm), and shorter excretory pore The new species belongs to the Goettingiana group distance from the anterior end (114 µm vs 130 µm). of Heterodera; currently, the group contains seven The new species differs from H. circeae having a valid species, viz, (Liebscher, smaller size of cysts (495 × 384 µm vs 555 × 397 µm), 1892), H. carotae (Jones, 1950), H. cruciferae (Franklin, a shorter fenestral length (31 µm vs 43 µm), vulval 1945), H. circeae (Subbotin and Turhan, 2004), H. slit length (39 µm vs 48 µm), longer J2s body length scutellariae (Subbotin and Turhan, 2004), H. urticae (568 µm vs 434 µm), stylet knobs (rounded and (Cooper, 1955), and H. persica (Maafi et al., 2006). slightly sloping posteriorly vs rounded and flat or The new species differs from H. goettingiana by slightly concave anteriorly), higher MB value (86 µm having a shorter fenestral length (31 µm vs 35 µm), vs 70 µm), longer excretory pore distance from the absence of bullae (vs few), weak underbridge (vs anterior end (114 µm vs 101 µm), longer tail length 117 µm), longer J2s body length (568 µm vs 486 µm), (57 µm vs 52 µm), and longer length of hyaline tail stylet knobs rounded and flat or slightly concave portion (29 µm vs 26 µm). anteriorly vs smoothly rounded to slightly hook- The new species differs from H. scutellariae, shaped with a recurved anterior surface, longer having smaller cysts (495 × 384 µm vs 560 × 424 µm), distance of median bulb from the anterior end (MB) by a shorter fenestral length (31 µm vs 35 µm), vulval (86 µm vs 70 µm), shorter excretory pore distance slit length (39 µm vs 43 µm), longer J2s body length from the anterior end (114 µm vs 158 µm), and shorter (568 µm vs 408 µm), higher MB value (86 µm vs 62 µm), length of hyaline tail portion (29 µm vs 37 µm). longer excretory pore distance from the anterior end The new species is differentiated from H. carotae (114 µm vs 89 µm), longer tail length (57 µm vs 47 µm), by having a bigger size of cysts (495 × 384 µm vs and longer length of hyaline tail portion (29 µm vs 408 × 309 µm), shorter vulval slit length (39 µm vs 25 µm).

7 Heterodera microulae sp. n. from China: Li et al.

4 5 31 39 25 26 86 57 29 568 114 Weak sp. n. Absent 495×384

H. microulae Microula sikkimensis China Rounded and flat or slightly concave anteriorly (Maafi et al., g g 4 5 35 86 43 21 24 62 89 47 25 408 Absent 560 × 424

H. scutellariae Scutellaria galericulata Germany Slightly convave anteriorly f 4 6 43 83 48 22 25 70 52 26 434 101 (Subbotin and Turhan, 2004); Absent e,f 555 × 397

group (all measurements are in µm). H. circeae Circaea lutetiana Germany Rounded and slightly sloping posteriorly e

– 4 8 38 46 23 27 58 29 541 130 Weak Absent 492 × 435

H. urticae Urtica dioica L. Northern Ireland Slightly concave anteriorly (Mathews, 1970); Goettingiana d d 4 6 47 49 23 23 70 47 24 104 440 103 Present 533 × 380

H. persica Heracleum persicum Desf. ex Iran Rounded or slightly projecting anterior, convex posteriorly c (Stone and Rowe, 1976); c – – 4 34 85 45 24 68 50 25 431 101 Absent 429 × 333

H. cruciferae Brassica oleracea L.V.capitata England Anterior face flat to concave b 4 31 90 47 21 24 66 99 52 28 5-6 422 (Mathews, 1975); b Absent 408 × 309 var. sativa England Concave anterior face H. carotae a 4 5 35 39 25 25 70 60 37 117 486 158 Few 521 × 372

H. goettingiana Pisum sativum L. Germany Smoothly rounded to slightly hooked shaped with recurved anterior surface (Stone and Course, 1974); a Vulval slit Body length a Stylet knobs Stylet length Lateral line Excretory pore Hyaline portion Species Host Locality Cyst Size Fenestral length Underbridge length Bullae length Second-stage juvenile DGO MB from anterior end Tail of tail length Table 2. Main morphological character of represent species from the Notes: Data from: 2006).

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Additionally, comparative morphological and mor­ 28S gene (Fig. 5) represented a highly supported phometric characters of H. microulae sp. n. with (posterior probability PP = 100) clade of Heterodera other valid species of Goettingiana group are listed species, where Goettingiana group species occupied in Table 2. a basal position. It is noted that H. microulae sp. n. clustered together with Heterodera sp. DP-2010 Molecular characterization and (HM560855, HM560856) from Qinghai, China and phylogenetic relationships forms a 100% supported clade. The ITS-rDNA sequence (accession no. MT573437) The H. microulae sp. n. sequences of D2-D3 region divergence of H. microulae sp. n. with other of 28 S (734 bp), ITS (993 bp), and COI (415 bp) gene Goettingiana group species is as follows: 0.20% were obtained and submitted to the GenBank. (2-bp difference), 0.4 to 0.5% (4-bp difference), The D2-D3 of 28S-rRNA sequence (accession 3.02% (29-bp difference), 5.01% (48-bp difference), no. MT573436) of H. microulae sp. n. showed 5.11% (49-bp difference), 7.45% (72-bp difference), 97.09% (19-bp difference), 97.66 to 98.49% (11-17-bp 6.77 to 6.95% (67-68-bp difference), 6.29 to 7.25% difference), 98.38% (9-bp difference), 98.62% (9-bp (66-70-bp difference), and 7.41 to 8% (74-77-bp difference), 98.45% (11-bp difference), and 99.86 to difference) for Heterodera sp. DP-2010 (HM560791), 100% (0-1-bp difference) sequence identities with H. goettingiana (HM370423, HM370425), H. persica H. goettingiana (DQ328697), H. carotae (KX463292 (AF498377), H. scutellariae (AY368995), H. circeae and KX463293), H. cruciferae (KP114546), H. urticae (AY368994), H. urticae (AF274412), H. carotae (DQ328696), Heterodera sp. RH-2010 (GU456692) (AF274413; MG976790), H. cruciferae (AF274411; from Iran, and Heterodera sp. DP-2010 (HM560856 GU126668), and H. goettingiana (KY129827; and HM560855) from Qinghai, China, respectively. AF274411; AF498374), respectively. The Bayesian The Bayesian phylogenetic tree of the D2-D3 of phylogenetic tree of the ITS gene (Fig. 6) represented

Figure 5: Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from 28S D2/D3 extension region under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *Identified as Heterodera sp. by Ye et al. (unpublished) and Peng et al. (unpublished) in the GenBank.

9 Heterodera microulae sp. n. from China: Li et al.

Figure 6: Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from ITS region under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *Identified as Heterodera goettingiana by Peng et al. (unpublished); **Identified as Heterodera sp. by Peng et al. (unpublished); ***Identified as Heterodera goettingiana by Huang et al. (unpublished) in the GenBank.

a highly supported (posterior probability PP = 100) of Heterodera species (HM560791; HM370425) with clade of Heterodera species. As in the 28S tree, 100% support. the ITS tree also positioned the Goettingiana The COI gene sequence of H. microulae sp. n. group species. H. microulae sp. n. (MT573437) showed 87.21 to 89.53% (differing from 36 to 44 bp), clustered with H. persica (AF498377), H. scutellariae 88.19% (differing from 43 bp), 88.67 to 88.92% (AY368994), H. circeae (AY368995), Heterodera (differing from 46 to 47 bp), and 88.67 to 89.40% sp. DP-2010 (HM560791), and H. goettingiana (differing from 44 to 47 bp), sequence identities with (HM370423, HM370425) from Qinghai, China with H. goettingiana (KY129829-KY129831), H. urticae high-probability support (pp = 91%). It is also noted (MK093155 and MK093156), H. cruciferae (MG563230 that sequences of H. goettingiana (HM370423, and MG563234), and H. carotae (KX463299- HM370425) from Qinghai, China, clustered outside KX463306, MG563227, MG563229, MG563231- with other H. goettingiana (KY129827, AF274411, MG563233, and MN820659), respectively. The and AF498374) subclades and should be considered Bayesian phylogenetic tree of the COI gene (Fig. 7) a misidentification. However, H. microulae sp. represented a highly supported (posterior probability n. (MT573437) is clustered with H. sp. DP-2010 PP = 100) clade of Heterodera species. In this tree, (HM560791) and H. goettingiana (HM370423, H. microulae sp. n. clustered with H. goettingiana, HM370425) from Qinghai, China, with 100% H. urticae, H. cruciferae, and H. caratae with 98% support. It is also noted that H. microulae sp. n. support; however, H. microulae sp. n. formed a (MT573437) clustered with two Chinese populations separate clade from those sequences.

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Figure 7: Molecular phylogenetic tree of H. microulae sp. n. (highlighted in bold) inferred from COI gene under GTR + I + G model. The posterior probability values exceeding 50% are given on appropriate clades. *KC172916 identified as H. pratensis by Toumi et al. (2013) and later corrected to by Madani et al. (2018).

Discussion Turhan, 2004). The new species also belong to the Goettingiana group and morphologically very close Taxonomy of Heterodera species has been revised to H. urticae; however, morphometrics of J2s body extensively in the past; Baldwin and Mundo- lengths, DGO and excretory pore position, fenestral Ocampo (1991) placed 23 Heterodera species length, vulval slit length, and cyst width can be used into Goettingiana group. However, Sturhan (1998) to differentiate both species. and Subbotin et al. (2001) used J2’s lateral field Phylogenetically, it is evident that H. microulae characters and host preferences to separate sp. n. is a member of Goettingiana group. In our Heterodera species into different groups (such as analyses, it is also noted that Heterodera sp. DP- Bifenestra, Cyperi, and Humuli groups). The key 2010 (HM560791, HM560855, and HM560856) and morphological characters of the Goettingiana group H. goettingiana (HM370423 and HM370425) from include lemon-shaped cysts having a protruding Qinghai, China, formed a well-supported molecular neck, ambifenestration, and absence of bullae (small clade with the H. microulae sp. n. Moreover, the bullae occasionally present); some species may have nucleotide differences of these sequences with an egg sac, vulval slit length > 35 µm, a thin vulval our new species sequences are also very low bridge, fenestral length (30-45 µm), and a weak (2-4-bp difference for ITS and 0-1 bp for 28S). underbridge. There were second-stage juveniles Previously, Escobar-Avila et al. (2018) indicated that with body length > 400 µm, stylet length > 20 µm, the sequences of H. goettingiana (HM370423 and tail length > 45 µm, hyaline tail portion > 20 µm, HM370425) from Qinghai, China, might be a case and lateral field with four lines (Subbotin and of misidentification. Based on our phylogenetic and

11 Heterodera microulae sp. n. from China: Li et al. sequence analysis results, we regard Heterodera sp. De Grisse, A. T. 1969. Redescription on modifications DP-2010 (HM560791, HM560855, and HM560856) de quelques techniques utilisees dans letude des and H. goettingiana (HM370423 and HM370425) as nematodes phytoparasitaires. Mededlingen Rijksfaculteit H. microulae sp. n. der Landbouwwetenschappen Gent, 34:351–69. Heterodera microulae sp. n. is isolated from De Ley, P., Tandingan De Ley, I., Morris, K., Abebe, Microula sikkimensis, it is a biennial herbaceous E., Mundo-Ocampo, M., Yoder, M., Heras, J., Waumann, plant that grows in forests, meadows, and forest D., Rocha-Olivares, A., Burr, A. H. J., Baldwin, J. G. and edges at altitudes of 2,200 to 4,700 m, and it is widely Thomas, W. K. 2005. An integrated approach to fast and informative morphological vouchering of nematodes distributed in South and East Asian countries (Pi et al., for applications in molecular barcoding. Philosophical 2014). H. microulae sp. n. was found in Gansu and Transactions of the Royal Society of London. Series B, Qinghai Provinces, but we speculate that it is likely to Biological Sciences 360:1945–58. be found in some localities that are characterized by Ding, Z., Namphueng, J., He, X. F., Peng, D. L. and low temperature, high rainfall, and high altitude. Huang, W. K. 2012. First report of the cyst nematode The present study described a new species found () on in Hunan Province, in the rhizosphere of M. sikkimensis; further research China. Plant Disease 96:151. is needed to understand the distribution and biology Escobar-Avila, I. M., Lopez-Villegas, E. O., of the new species. In addition, plenty of leguminous Subbotin, S. A. and Tovar-Soto, A. 2018. First report crops (pea, kidney bean, pole bean, etc.) are growing of cyst nematode Heterodera carotae in Mexico: in the same locality. Therefore, host-suitability tests morphological, molecular characterization, and host of H. microulae sp. n. are an open research field to range study. Journal of Nematology 50:229–42. investigate the damage potential of this species. Franklin, M. T. 1945. On n. sp. of Brassicas, and on a Heterodera strain infecting Clover and Dock. Journal of Helminthology 21:71–84. Acknowledgments Golden, A. M. 1990. “Preparation and mounting This research was supported by the National Natural nematodes for microscopic observations”, In Science Foundation of China No. 31760507 and the Zuckerman, B. M., Mai, W. F. and Krusberg, L. R. Special R & D Fund for Public Benefit Agricultural, (Eds), Plant Nematology Laboratory Manual University of Massachusetts Agricultural Experiment Station, No. 201503114. The authors thank the assistance Amherst, MA, 197–205. of the Institute of Plant Protection of China for the Handoo, Z. A. and Subbotin, S. 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Table S1. Goettingiana group species, locality, host plants, and GenBank accession number used in this study.

Accession Species Locality Host-plant Marker number

H. goettingiana Germany Pisum sp. ITS AF274414 H. goettingiana Lorestan, Doroud, Akbar Abad, Iran Trifolium repens ITS AF498374 H. goettingiana Monopoly, Bari province, Italy Pisum sativum ITS KY129827 H. sp Yuekou village, Tianmen county, Hubei – ITS HM560794 province, China H. sp Morroco – ITS AY347918 H. carotae Creances, France Daucus sp. ITS AF274413 H. carotae South Africa Daucus carota ITS MG976790 H. cruciferae Brielle, The Netherlands Brassica sp. ITS AF274411 H. urticae Diksmuide, Belgium Urtica sp. ITS AF274412 H. cruciferae Moscow, Russia Brassica oleracea ITS GU126668 H. sp Xinzhuang village, Huangzhong – ITS EU623623 county, Qinghai province, China H. microulae sp. n. Tianzhu county, Gansu province, Microula sikkimensis ITS MT573437 China H. microulae sp. n. Haiyan county, Qinghai province, – ITS HM560791 China H. microulae sp. n. Haibei city, Qinghai province, China – ITS HM370425 H. microulae sp. n. Xining city, Qinghai province, China – ITS HM370423 H. persica Tehran, Dizin, Iran Heracleum persicum ITS AF498377 H. scutellariae Bremen, Germany Circaea lutetiana ITS AY368994 H. circeae Muenster, Germany Scutellaria galericulata ITS AY368995 H. carotae Ontario province, Canada Daucus carota 28S KX463292 H. carotae Ontario province, Canada Daucus carota 28S KX463293 H. sp Yuekou village, Tianmen county, Hubei – 28S HM560857 province, China H. sp Iran – 28S GU456692 H. cruciferae Iran – 28S KP114546 H. urticae Belgium – 28S DQ328696 H. goettingiana Iran – 28S DQ328697 H. microulae sp. n. Haiyan county, Qinghai province, – 28S HM560855 China H. microulae sp. n. Haomen village, Menyuan county, – 28S HM560856 Qinghai province, China H. microulae sp. n. Tianzhu county, Gansu province, Microula sikkimensis 28S MT573436 China H. carotae South Africa Daucus carota COI MN820659 H. carotae Mesola, Forli-Cesena province, Italy Daucus carota COI KX463299 H. carotae Mesola, Forli-Cesena province, Italy Daucus carota COI KX463300

15 Heterodera microulae sp. n. from China: Li et al.

Accession Species Locality Host-plant Marker number

H. carotae Margherita, di Savoia, Italy Daucus carota COI KX463301 H. carotae Margherita, di Savoia, Italy Daucus carota COI KX463302 H. carotae Ontario province, Canada Daucus carota COI KX463303 H. carotae Ontario province, Canada Daucus carota COI KX463304 H. carotae Ontario province, Canada Daucus carota COI KX463305 H. carotae Ontario province, Canada Daucus carota COI KX463306 H. carotae Mexico Daucus carota COI MG563227 H. carotae Mexico Daucus carota COI MG563229 H. carotae Switzerland – COI MG563231 H. carotae Switzerland – COI MG563232 H. carotae France – COI MG563233 H. carotae Belgium – COI KC172916 H. urticae Faulkner county, Arkansas, USA Stellaria media COI MK093155 H. urticae Faulkner county, Arkansas, USA Stellaria media COI MK093156 H. cruciferae California, USA – COI MG563230 H. cruciferae Moscow region, Russia – COI MG563234 H. goettingiana Monopoly, Bari province, Italy Vicia faba COI KY129829 H. goettingiana Monopoly, Bari province, Italy Pisum sativum COI KY129830 H. goettingiana Monopoly, Bari province, Italy Medicago lupulina COI KY129831 H. microulae sp. n. Tianzhu county, Gansu province, Microula sikkimensis COI MT576084 China Note: Newly added sequences are indicated by bold font.

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